Cell fate specification occurs through the tightly regulated expression of key transcription factors at precise levels, times, and places during development. The expression of these determinants is controlled by enhancers and promoters. This proposal focuses on two cases where the level and timing of gene expression are especially important for cell fate specification in the Drosophila visual system. A number of mutations have been shown to cause abnormal gene regulation and cell fate, making a better understanding of these processes highly relevant to understanding the genetic basis of disease. Live imaging of stochastic gene activation in the visual system: In Aim 1, I will examine the stochastic specification of cell fate in the Drosophila retina, where otherwise equivalent R7 photoreceptor precursors have a particular probability of taking one of two fates important for color vision. This decision is controlled by the stochastic, cell-intrinsic expression of the transcription factor Spineless. I will examine two possible sources of spineless stochasticity: variability in transcriptional activation at the level of the promoter, and variation in chromatin accessibility. In order to quantify the underlying transcriptional dynamics, I will image gene expression in real time using the MS2 and PP7-based live transcriptional imaging systems. MS2 live imaging has recently revolutionized the study of gene regulation in the Drosophila embryo by providing a new level of quantitative measurement. This approach combined with direct tagging by CRISPR/Cas9 of the spineless locus will allow me to determine the origin of stochasticity in this cell fate decision. Factors that influence stochastic fate: In Aim 2, I will directly test two models that predict how different factors influence the probability of spineless expression. To directly test the role of transcriptional initiation vs. chromatin state, I will modify the spineless basal promoter using CRISPR/Cas9 by replacing it with characterized promoters that have been shown to initiate expression more or less robustly in the Drosophila embryo, for instance through recruitment of paused RNA Polymerase II. This will allow me to test the role of the promoter in stochastic fate decisions. If stochastic outcomes are unaffected by such changes, I will test the role of local chromatin state via local, targeted changes in chromatin state to evaluate the effect on the stochastic ratio of fates produced. This approach will be complemented by live imaging of transcription, which will provide an additional means of assessing the quantitative effects of specific modifications. Studying the role of transcriptional timing and levels in temporal transitions in neuroblast fate: Aim 3 will examine a series of temporal transitions between five different transcription factors in medulla neuroblasts that generate neural diversity in the fly brain. Although we know that there is cross regulation among these temporal transcription factors, the mechanisms that time these transitions are still unknown. I will test the influence of the level and variability of expression of individual factors on the timing or output of each temporal window. MS2 imaging used in combination with fluorescent protein tags will enable direct comparison between expression levels of one temporal factor with the initiation of transcription of the next factor in the series. This will provide insight into mechanisms that govern the timing the temporal windows and their transitions. A CRISPR-based approach will then be used to modify the amount of activation of individual transcription factors in the temporal series to test whether transcriptional levels or variability play a role in controlling the duration of the temporal window. Understanding how gene regulation influences cell fate will lead to a better understanding of how cell fate is determined and maintained. Ultimately, I hope that this will lead to new strategies for programming or reprograming cell fates in a purposeful way. A K99 Transition Award would allow me to receive additional training in quantitative imaging methods and provide time for tool development for this novel approach to studying stochastic fate regulation and temporal fate specification. The training phase of this proposal will be performed in the laboratory of my mentor Dr. Claude Desplan. I can think of no better place to study exciting and engaging questions in the development of the Drosophila visual system, or for tool development and collaboration with insightful colleagues. The NYU Center for Developmental Genetics, Biological Imaging Facility, and neighboring Center for Genomics and Systems Biology provide the facilities and equipment necessary for the proposed work. I have thoroughly enjoyed the challenges and excitement of academic research in graduate school and as a postdoc. These experiences, and the opportunity to share them with others, have encouraged me to plan my career with the goal of establishing an independent academic research laboratory.